Apoptosis or programmed cell death is a biological process critical for normal development and cellular homeostasis. Its abnormal occurrence has been implicated in a number of disorders including stroke and neurodegenerative diseases. The translocation of cytochrome c (Cyt c) from mitochondria is a key event in the initiation or amplification of apoptosis and may be a suitable target for drug intervention in apoptosis-related maladies. Thus, understanding the mechanism of Cyt c release is of supreme importance. In the proposed studies, monoclonal antibodies (mAbs) will be prepared against central nervous system (CNS) mitochondrial outer membrane (OM) components in an attempt to identify novel proteins involved in Cyt c translocation. The mAbs will be tested for inhibition of BID/BAX-induced release of Cyt c from isolated rat brain mitochondria using a capture enzyme-linked immunosorbent assay to measure Cyt c. The antigen recognized by any inhibitory mAb will be identified by immunoprecipitation and protein microsequencing. Using reversible chemical cross-linking reagents, attempts will be made to couple Cyt c to OM components it may contact as it translocates through the OM in response to the pro-apoptotic proteins BID and BAX. Anti-Cyt c mAbs already prepared in this laboratory will be used to immunoprecipitate Cyt c complexes and any associated molecules will be identified by microsequencing after electrophoresis of the de-cross-linked complexes in polyacrylamide gels. mAbs against a variety of OM antigens will likely arise in the first study, some of which may identify or more clearly define additional communication networks between mitochondria and other cellular organelles. The feasibility of the proposed research is supported by preliminary results and by the participation of on-site collaborators with expertise in the field of neuroscience, in particular the physiology of CNS mitochondria.